Real-Time QCM-D Immunoassay through Oriented Antibody Immobilization Using Cross-Linked Hydrogel Biointerfaces

Abstract

This report presents the development of pre-cross-linked and in situ cross-linked polyethyleneimine-carboxymethylcellulose antibody immobilization platforms for real-time QCM-D immunoassay of sepsis-related biomarkers. These platforms differ significantly from recent trends in QCM-based assays, a rapidly expanding field given the affordability and sensitivity of the transduction system, by providing ultrafast biointerface deposition through cross-linking of polysaccharides. Using rhIL-1ra (17 kDa), a known sepsis biomarker, for development, various immunoassay modifications to increase sensitivity were investigated, including the use of Protein A, Protein G, and anti-IgG Fc specific antibody capture ligands for oriented antibody immobilization, higher-frequency QCM-D crystals, and amplification using secondary antibodies. The optimized assay employs Protein A oriented immobilization on pre-cross-linked polymer and secondary antibodies to achieve a detection limit of 25 ng/mL on 5 MHz crystals. Assay repeatability using the optimized chemistry is robust, with no loss in 100 ng/mL antigen detection over 20 cycles of the 10 min sandwich assay. Nonspecific adsorption of human serum albumin, as characterized by ToF-SIMS, is minimal and negligible for the pre-cross-linked and in situ cross-linked compositions, respectively.

Surface Functionalized Nanofibrillar Cellulose (NFC) Film as a Platform for Immunoassays and Diagnostics

“…Moreover, the negligible non-specific adsorption of human IgG on NFC surface after the superblock treatment allows the use of NFC films in Biointerphases (2012) immunoassays, where positively charged target molecules are monitored. The amount of immobilized antihuman IgG on activated NFC-film was found to be similar to those reported in the literature [5,32]. It should also be mentioned that the adsorbed amount of human IgG on the antihuman IgG interface was over twofold when compared to published results with anti-hIL-1ra conjugation on CMC-PEI hydrogel [5].…”

“…The amount of immobilized antihuman IgG on activated NFC-film was found to be similar to those reported in the literature [5,32]. It should also be mentioned that the adsorbed amount of human IgG on the antihuman IgG interface was over twofold when compared to published results with anti-hIL-1ra conjugation on CMC-PEI hydrogel [5].…”

Orientation and characterization of immobilized antibodies for improved immunoassays (Review)

“…EDC/NHS chemistry has been employed as a covalent attachment methodology for immobilization of proteins and also as a method for the preparation of substrates. Carrigan et al 51 used EDC/NHS in two ways, (1) for the cross-linking of polyethylenimine and carboxymethyl cellulose, and (2) for activation of this substrate for protein binding targeting amine and carboxyl functionality. Sun et al 52 took a novel approach to EDC/NHS coupling by introducing NHS reactive groups to random sites on an antibody then using an electric field to preferentially orient the antibody, via its intrinsic dipole, before reaction to the free amines on cysteine immobilized to a gold electrode.…”

“…51,158 Via this method, protein orientation may be investigated; for instance, an antibody immobilized close to the surface in a flat-on orientation will have a low viscoelastic dissipation, while in contrast, an antibody in head-on or end-on orientation will have a higher viscoelastic dissipation.…”

A Reusable Capacitive Immunosensor Based on a CuS Ultrathin Film Constructed by Using a Surface Sol-Gel Technique

“…However, a label-free immunosensor 6,7 has provoked more interest for many reasons, such as simplicity and real-time compared with the traditional immunoassay technology, like enzyme-linked immunosorbent assay (ELISA) 8 and radioimmunoassay. 9 Among those immunosensors, the capacitive immunosensor has attracted more interest with high sensitivity, rapid test time and common instrumentations [10][11][12][13][14][15] compared with other types of direct biosensors, such as surface plasmon resonance, 16,17 quartz crystal microbalance (QCM), 18 surface acoustic wave, 19 and surface scanning detection. 20 The fabrication of a biocompatible layer is more pivotal to a capacitive immunosensor.…”